Growth without growth hormone: can growth and differentiation factor 5 be the mediator?

Growth without growth hormone (GH) is often observed in the setup of obesity; however, the missing link between adipocytes and linear growth was until now not identified. 3T3L1 cells were induced to differentiate into adipocytes and their conditioned medium (CM) (adipocytes CM, CMA) was added to metatarsals bone culture and compared to CM derived from undifferentiated cells. CMA significantly increased metatarsals bone elongation. Adipogenic differentiation increased the expression of growth and differentiation factor (GDF)-5, also found to be secreted into the CMA. GDF-5 significantly increased metatarsal length in culture; treatment of the CMA with anti-GDF-5 antibody significantly reduced the stimulatory effect on bone length. The presence of GDF-5 receptor (bone morphogenetic protein receptor; BMPR1) in metatarsal bone was confirmed by immunohistochemistry. Animal studies in rodents subjected to food restriction followed by re-feeding showed an increase in GDF-5 serum levels concomitant with nutritional induced catch up growth. These results show that adipocytes may stimulate bone growth and suggest an additional explanation to the growth without GH phenomenon.


Introduction
While normal growth in children is usually controlled by the growth hormone (GH)-insulin-like growth factor (IGF)-1 axis, it has been recognized that growth can occur also in its absence, especially in clinical situations that involve obesity. Obese children grow at a normal rate despite their low serum GH levels; children with hypopituitarism secondary to craniopharyngioma resection may continue to grow and may even show growth rate acceleration if their weight increases significantly (Geffner, 1996;Lazar et al., 2003;Phillip et al., 2002).
Adipocytes are not only the main site for energy storage, releasing fatty acids when required, but also an exceptionally active secretory tissue, releasing many endocrine and paracrine factors, commonly referred to as adipokines, which affect both peripheral tissues and the central nervous system. Adipokines are involved in diverse physiological processes, including immune response (Pond, 2005), energy homeostasis (Havel, 2002) and bone growth (Berendsen & Olsen, 2014;Maor et al., 2002).
The aim of the present study was to examine the relationship between adipocytes and bone growth and to identify potential mediators of this association. To test this hypothesis we used an in vitro model of 3T3L1 cells induced to differentiate in culture into adipocytes, and metatarsals bone rudiments. The 3T3-L1 pre-adipocyte cell line was originally isolated for its ability to undergo differentiation into adipocytes in vitro. It is now the predominant model for the study of adipogenesis (Green & Kehinde, 1975;Green & Meuth, 1974). DNA microarray studies have been carried out to investigate specific cellular programs in the regulation of gene expression during differentiation of 3T3L1 pre-adipocytes. Soukas et al. (2001) reported that the levels of expression of 1259 transcripts changed threefold or more during 3T3L1 differentiation, and in a similar study, Burton et al. (2004) identified 636 transcripts that were up-regulated at least twofold and 380 transcripts that were down-regulated in adipocytes compared to pre-adipocytes (Burton et al., 2004). Recently, researchers using a 5-plex stable isotope labeling by amino acids in cell culture (SILAC)-based strategy described a temporal profile of nuclear and secreted proteins during adipocyte differentiation (Molina et al., 2009).
The results of our study show that the conditioned medium (CM) of 3T3L1 adipocytes (CMA) stimulates the growth of metatarsal rudiment bones in culture and that growth and differentiation factor (GDF)-5 is the principal mediator in this setting. This finding adds another nutrition-induced growthstimulating factor to the currently known battery.

Morphological studies
Metatarsals were fixed in 10% neutral buffered formalin, embedded in paraffin and 6 mm paraffin sections were produced using a microtome (Leica RM2145) from the center of the bones. Morphological staining was performed using Alcian blue, hematoxylin and eosin, as follows: deparaffinization in o-xylene for 20 min, dehydration in 100% ethanol followed by 95% ethanol, 10 min each, followed by a 5-min wash with tap water. Slides were incubated for 3 min in 3% acetic acid and then in Alcian Blue (pH 2.5) for 30 min. After 10 min of washout by tap water, the slides were incubated with hematoxylin stain for 8 min and washed with tap water for 10 min. Eosin stain was added to the slides for 2 min to complete the staining. Slides were photographed with the Olympus DP71 camera.
The hypertrophic zone height, the number of hypertrophic cells per column, the height of hypertrophic cells and the length of the ossification zone were measured using the Image ProPlus software (version 4.5.1.22, Media Cybernetics), essentially as described (Chagin et al., 2010). Two sections of 10-12 bones from each group were analyzed.

Immunohistochemistry
For immunohistochemistry analysis, we used 6 mm sections of the paraffin-embedded metatarsals. The slide-mounted sections were deparaffinized with o-xylene and rehydrated with a graded series of ethanol. Endogenous peroxidases were neutralized with 3% hydrogen peroxide diluted with methanol, for 25 min. The slides were then incubated with a blocking agent (10% non-immune goat serum) for 20 min to block nonspecific binding of the primary antibody, followed by incubation with the primary specific antibody (GDF-5 receptor, BMPR1B) for 1.5 h and then washed three times with PBS. The negative control sections were incubated with non-immune rabbit serum (#08-6199; Zymed). Slides were incubated for 10 min with an enhanced biotinylated secondary antibody that recognizes the primary antibody, and washed three times with phosphate buffered saline (PBS). Enhanced horseradish peroxidase-conjugated streptavidin (HRP-SA) conjugated with aminoetyl carbazole as a substrate was added for another 10 min to allow binding to the biotinylated secondary antibody (Histostatin-SP kit, Zymed Laboratories -Invitrogen, Waltham, MA). Counterstaining was performed with hematoxylin followed by PBS washout. All steps were performed at room temperature.

Induction of 3T3-L1 cell differentiation into adipocytes
3T3-L1 cells were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% adult bovine serum (ABS), 4 nM glutamine, 100 U/ml penicillin, 100 mg/ ml streptomycin and 250 mg/ml amphotericin in a 5% CO 2 humidified atmosphere (37 C) to confluence on 100 mm plates. Two days after confluence, cells were treated with 10 mM dexamethasone, 0.25 IU/ml insulin and 0.5 mM 1methyl-3 isobutylmethylxanthine (IBMX) (differentiation mix) in DMEM supplemented with 10% FBS for 48-72 h followed by 0.25 IU/ml insulin alone in DMEM supplemented with 10% FBS for additional 48-72 h to induce adipocyte differentiation (Somjen et al., 1997). CM was collected after additional 6 days (at day 5 the medium containing 10% FBS was replaced by medium containing 1% or 5% of FBS) (see Supplementary Figure 1). Undifferentiated cells were grown in DMEM supplemented with 10% ABS until confluence. One day prior to cell harvesting the medium was replaced by medium containing 1% or 5% of FBS; both CMA and CMFcontain 5% FBS. Floating cells were eliminated by centrifugation, and the CM was filtered through a 0.22-mm filter and stored at À70 C until use.

Adipocyte staining
Cells were washed three times with PBS and then fixed with 4% paraformaldehyde for 20 min. Oil red O (0.5%) was prepared in isopropanol, mixed with water in a 3:2 ratio, and filtered through a 0.45-mm filter. The fixed cells were incubated with the Oil red O reagent for 30 min at room temperature and washed with water. The stained fat droplets in the cells were visualized under light microscopy and photographed (Supplementary Figure 1) (Molina et al., 2009).

Biochemical analysis of the CM
Free fatty acid content, total protein content and glucose levels in the CMs were measured with the Immulite 2000 (Siemens AG, Munich, Germany).

Mouse growth factor PCR array
Total RNA was extracted from the cells according to the acid guanidinium thiocyanate method (Chomczynski & Sacchi, 1987). The quantity and quality of the RNA were evaluated using a NanoDrop spectrophotometer (NanoDrop Technologies; Thermo Scientific, Wilmington, DE). Equal amounts of RNA from the 3T3L1 cells before and after differentiation were analyzed with the Mouse Growth Factor RT 2 ProfilerÔ PCR Array (PAMM-041A, SaBiosciences, Qiagen, Gaithersberg, MD), which profiles the expression of 84 genes related to growth factors. The panel includes probes for angiogenic growth factors, regulators of apoptosis, and genes involved in cell differentiation, embryonic development, and tissue-specific development. These include probes for the genes encoding members of the bone morphogenetic proteins (BMP) family, fibroblast growth factors (1-22), epidermal growth factor, insulin like growth factors 1 and 2, interleukins, growth and differentiation factors 5,10 and 11, leptin and others (see Supplementary Table 1). First-strand cDNA synthesis was performed with the RT 2 FirstStrand Kit (SaBiosciences) using 1 mg of total RNA. This step was preceded by DNase I treatment. Gene expression was measured by qPCR, according to the manufacturer's instructions, with the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Results were confirmed by qPCR reactions performed with specific RT 2 quantitative PCR primer assay-SYBR Green mouse GDF-5; Hprt1 served as the internal control, as of the five housekeeping genes presented on the array, it showed the least variations (PPM04450A and PPM035596, respectively, SaBiosciences). The following thermal cycling conditions were used: one cycle at 50 C for 2 min and at 95 C for 10 min, followed by 40 cycles of 15 s at 95 C and 1 min at 60 C. Relative expression was determined using the 2 ÀDD Ct method (Livak & Schmittgen, 2001). Each sample was examined in triplicate.

GDF-5 analysis
GDF-5 was measured in the different CMs, and animal serum using ELISA kits (for the CMs and mouse serum: CSB-EL009349MO, Cusabio Biotech, Hubei, P.R. China; for rat serum: SEC101Ra, Cloud-Clone Corp., Houston, TX) according to the manufacturer's instructions. The sensitivity of the assays as reported by the manufacturers are 9.75 and 55 pg/ ml, respectively.

Stable isotope labeling by amino acids in cell culture (SILAC)
SILAC followed by treatment on ProteoMiner Õ columns (BioRad, Hercules, CA) was used essentially as described by Molina et al. (2009) Once full isotope incorporation has been determined, cells were induced to differentiate. Subsequent SILAC analysis was performed on Tandem mass spectrometry (MS/MS) (The Weizmann Institute of Science, Rehovot, Israel).

Animal studies
Male Sprague-Dawley (SD) rats or ICR mice, 24 days old, were purchased from Harlan (Jerusalem, Israel) and housed individually at the animal care facility of the Felsenstein Medical Research Center. All animals had unlimited access to water. Animals were given 60% of the amount usually consumed by these animals at this age (Even-Zohar et al., 2008;. The food restriction was maintained for 10 days. At that point, animals were divided into two groups: one was kept restricted (RES, n ¼ 6), and the other was given normal chow ad libitum (that induced catch up growth, hence this group was named CU group; n ¼ 6). After 1 day of re-feeding, animals from the two groups were sacrificed by CO 2 inhalation; serum was collected and stored at À80 C until further use. Throughout the study, animals were observed daily, and all remained bright, alert and active, with no evidence of any disorder. The Tel Aviv University Animal Care and Use Committee approved all procedures.

Statistical analysis
Studies with CMA and CMF were performed five times. Each experiment on metatarsals was performed at least twice. Results are expressed as mean ± SEM. Student's T test or oneway analysis of variance (ANOVA) with Tukey post hoc were used to test for significant differences between the groups. The significance level was set at p50.05. SPSS v15 software (SPSS Inc., Chicago, IL) was used for statistical analysis of the data.

Effect of CM from adipocytes versus pre-adipocytes on metatarsal growth
The 3T3L1 cells were induced to differentiate with IBMX, dexamethasone and insulin (differentiation mix). Oil red O staining revealed substantial lipid accumulation (Supplementary Figure 1) (Somjen et al., 1997). After 6 days in culture, CM was collected, centrifuged, and filtered, and the supernatant was added in a 1:1 ratio to the metatarsals for up to 14-16 days, with a change of medium every other day. Metatarsal rudiment bones were grown in MEM containing 1% FBS and an equal volume of CM from undifferentiated 3T3L1 cells (fibroblasts; CMF) or differentiated 3T3L1 cells (adipocytes; CMA). A control group cultured in the presence of metatarsal medium alone was always included. Bone length increased significantly in response to CMA compared to CMF from day 8 onwards (p50.005; Figure 1). Biochemical analysis of the CM showed an increase in triglyceride content in CMA compared to CMF but no differences in total protein content, or glucose levels were noted (glucose in the range of 84-70 mg/dl, protein 0.3 g/dl in both, triglycerides 146 mg/dl in CMA and 100 mg/ dl in CMF).

Effect of leptin inhibitor on metatarsal growth in CMA
To determine if the growth-stimulating factor secreted by adipocytes is leptin, 2 mg/ml of anti-leptin was added to metatarsals grown in the presence of CMA. This concentration was calculated to be several hundred folds higher than the amount of leptin previously reported to be secreted by 3T3-L1 adipocytes (Norman et al., 2003). No significant effect on growth was observed (relative length increase: 80.97 ± 1.05% for CMA and 84.66 ± 2.26% for CMA+ leptin inhibitor; p ¼ 0.168).

Identification of the growth-promoting factor
The growth conditions of the adipocytes had a significant effect on the purification. Normally, adipocytes are grown in the presence of 10% FBS. To achieve better purification of the growth factor, we reduced the content of FBS in the medium. Interestingly, whether the concentration of FBS was 5% or 1%, the cells maintained their morphological appearance with no apparent cell death. However, CMA from cells grown with 1% FBS was significantly less effective in supporting growth than the CMA from cells grown with 5% FBS, with findings essentially similar to those of CMF (data not shown).
Use of SILAC followed by ProteoMiner Õ columns to purify the relevant protein (Molina et al., 2009), proved unsuccessful, as the best effect on metatarsals' growth was achieved when the adipocytes were grown in the presence of 5% FBS. Under these conditions, the level of serum albumin was so high that even after treatment with the ProteoMiner Õ and subsequent gel purification, significant amounts of albumin were present in the extract and hindered the MS/ MS analysis of the less abundant labeled proteins.

Expression array analysis
We therefore decided to take a molecular approach, using an expression array of growth factors. The results showed an increase in the expression of 19 growth factors in the adipocytes compared to the non-differentiated 3T3L1 cells (the 10 most affected growth factors are shown in Table 1). Given that GDF-5 was the most highly affected factor and that GDF-5 was previously shown to affect the growth and differentiation of skeletal elements (Buxton et al., 2001), we selected it as the focus of our studies.
The results were confirmed with qPCR using a GDF5specific probe set compared to housekeeping gene Hprt1. GDF5 expression was significantly increased by differentiated adipocytes compared to non-differentiated cells (results of real-time PCR showed 74-fold increase; p50.05; average Ct in fibroblasts was 33.36, average Ct in adipocytes 26.2; CT of Hprt1 -fibroblast -23.25; adipocytes: 22.3). Technical replicates were used for the qPCR, and biological replicates were used for the array and qPCR.

Effect of GDF-5 on metatarsal growth
Quantitative analysis of the GDF-5 content of CM revealed that the level of GDF-5 in CMF was below the detection limit (which is reported to be 9.75 pg/ml) of the ELISA kit. The level in the CMA was in the range of 100 pg/ml. We added GDF-5 directly to the culture medium of the metatarsals bone rudiments and followed the effect on bone elongation for several days (10, 100 and 1000 pg/ml; Figure 2). The results showed that GDF5 at concentrations of 100 and 1000 pg/ml significantly stimulated bone growth compared to controls, with maximal effect achieved already at a concentration of 100 pg/ml (which is the level of GDF5 in the CMA). The effect was evident from the beginning of the culture, but was statistically significant from day 6 onwards (Figure 2b; p at the last day 50.0005).
Histological examination of the bones showed that the most affected region was the hypertrophic zone ( Figure 3). The height of the hypertrophic zone, the number of cells in each column and the height of the hypertrophic cells were all increased by GDF-5 treatment.
To confirm that the growth stimulating effect is mediated by GDF-5, we incubated CMA with 50 ng/ml of anti-GDF-5 antibody for 2 h at RT (this concentration is 500-fold higher than average GDF-5 level in CMA). CMA was then centrifuged, filtered and separated into aliquots. The growth of metatarsals that were incubated with anti-GDF-5 treated CMA was significantly reduced compared to metatarsals grown in the presence of untreated CMA, and was not significantly different from that of the CMF (Figure 4). Metatarsal rudiment bones were grown in MEM containing 1% FBS and an equal volume of CM from undifferentiated 3T3L1 cells (fibroblasts; CMF, marked as triangles) or differentiated 3T3L1 cells (adipocytes; CMA, marked as diamonds). The cell cultures were grown for the last 24 h prior to medium collection in 5% FBS. A significant increase in bone length was found in response to CMA (CMA vs. CMF *p50.05; **p50.005). Relative length increase was calculated as the percentage of day ''0'', the first day of the experiment, results presented are mean ± SEM.

Immunohistochemistry of BMPR1
The presence of the receptor for GDF-5, BMPR1B (Nickel et al., 2005), on metatarsal bone was confirmed by immunohistochemistry ( Figure 5). Most of the staining was found in the resting and hypertrophic cells as well as in the perichondrium.

GDF-5 in nutritional induced catch up growth in animal models
The body weight of the food restricted animals was 18.6 ± 2.0 and 57.04 ± 3.96 g for mice and rats, respectively; refeeding led to a rapid increase in weight (CU mice 25.28 ± 1.025 g, CU rats 81.77 ± 6.53 g; p50.001 between RES and CU groups in each species). Analysis of serum levels of GDF-5 in these animals showed a significant increase in GDF-5 in serum derived from re-fed animals (CU), compared to the level in the RES groups in both mice and rats ( Figure 6).

Discussion
Our study clearly shows that adipocyte-secreted GDF-5 directly stimulates metatarsal bone growth in vitro. These findings may suggest an additional link between adipose tissue and longitudinal growth, implied by findings of growth without GH (Geffner, 1996;Lazar et al., 2003;Phillip et al., 2002), occurring mostly in the presence of hyperphagia and obesity.
To investigate the direct connection between adipocytes and growth, we used the metatarsal bone model (De Luca et al., 2000). In this manner, we were able to measure the effects on the length of the long bones in culture while maintaining the cells in their cartilaginous environment, including the extracellular matrix (collagens, proteoglycans, etc.) as well as the interactions between the cells in their different differentiation steps (quiescent, proliferative and hypertrophic zones).
Leptin, known to be secreted by adipocytes, was previously shown by us as well as others to directly stimulate chondrocyte and bone growth (Ducy & Karsenty, 2000;Gat-Yablonski et al., 2009;Maor et al., 2002;Pelleymounter et al., 1995;Turner et al., 2013). As leptin is secreted by adipocytes, it was our first growth promoting factor candidate, however, using a leptin inhibitor in a concentration of at least 670-fold higher than that secreted by 3T3L1 cells (Norman et al., 2003), we ruled out the possibility that leptin serves as the mediator in this context. These results are supported by the studies of Molina et al. (2009) who also failed to find leptin in the secretome of 3T3L1 and of Norman et al. (2003) who reported 3 ng/ml of leptin in CM of these cells, a concentration that is much lower than that used in our previous in vitro studies [50 ng/ml (Ben-Eliezer et al., 2007) or 500 ng/ml (Maor et al., 2002)].
The growth/differentiation factors (GDFs) are a subfamily of the highly conserved group of bone morphogenetic factors GDF5 (pg/ml) ** * Figure 2. Dose effect of GDF5 on metatarsal growth. (a) GDF5 was added to the culture medium of metatarsals in concentrations of 0, 10, 100 and 1000 pg/ml and bone growth was monitored (relative to control); results are from the last day only. (b) GDF5 was added to the culture medium of metatarsals in physiological concentration (100 pg/ml) and bone growth was monitored for 16 days. Squares -GDF5, diamonds -control (*p50.05, **p50.005, ***p50.0005). Relative length increase was calculated as the percentage of day ''0'', the first day of the experiment; results presented are mean ± SEM. Table 1. Growth factors which expression was significantly increased by adipocyte differentiation (the 10 most affected growth factors are presented).
GDF-5, also termed cartilage-derived morphogenetic protein (CDMP) or BMP14, is active during mesenchymal cell condensation, initiating the initial stages of chondrogenesis by promoting cell adhesion (Buxton et al., 2001) and it can increase the size of skeletal elements (Buxton et al., 2001). GDF-5 was also shown to stimulate proteoglycan production in chondrocyte-like cells, leading to an increase in aggrecan and type II collagen gene expression and increased production of proteoglycans (Erlacher et al., 1998). In a large multinational genetic study, a locus near the GDF5 gene on  Days chromosome 20 was found to be associated with final height in humans (Miyamoto et al., 2007). In addition, a specific SNP was found to be associated with increased risk for osteoarthritis (Hinoi et al., 2014a;Reynard et al., 2014).
A deficiency of GDF5 has multiple effects on skeletal tissues, and mutations in the GDF5 gene were shown to cause chondrodysplasia and several types of brachydactyly (Al-Qattan et al. 2015;Everman et al., 2002;Faiyaz-Ul-Haque et al., 2002), acromesomelic chondrodysplasia and DuPan syndrome (Douzgou et al., 2008) in humans, as well as brachypodism in mice. The best characterized experimental model is the GDF-5-deficient brachypod mouse, which has . GDF-5 serum levels in food restricted or re-fed SD rats (A) or ICR mice (B). Twenty-four-day-old animals were subjected to 10 days of 40% food restriction. After 10 days, half of the animals were kept restricted (RES) and half were allowed to re-feed with no restriction for one day (CU).
shortened limb bones and reduced growth (Storm et al., 1994) associated with significantly longer phase duration of hypertrophy (Mikic et al., 2004). Over-expression of GDF-5 in mice led to chondrodysplasia with expanded primordial cartilage, which consisted of an enlarged hypertrophic zone and a reduced proliferating zone, not only in the limbs, but also in the axial skeleton (Tsumaki et al., 1999). GDF-5 was reported to increase the number of chondro-progenitor cells and accelerate chondrocyte differentiation to hypertrophy, essentially as shown here. The histological examination of the bones presented in the current study showed a significant increase in the length of the bones exposed to GDF-5, a significant increase in the hypertrophic cell number and height and the overall length of the hypertrophic zone, with agreement with previous publications (Coleman & Tuan, 2003;Mikic et al., 2004).
GDF-5 binds specifically to BMPR1B, BMPR2 and ACTR2a, forming a hetero-dimeric complex (Chen et al., 2006). We have shown that BMPR1B is present in the metatarsals bones, specifically in the hypertrophic zone, with agreement to its site of action.
GDF-5 was recently found to be expressed during the in vitro differentiation of 3T3L1 cells (Pei et al., 2014) and in vivo during the differentiation of brown and white adipocyte tissue (Hinoi et al. 2014a,b,c). Accordingly, we have found that GDF-5 was secreted to the CMA in a concentration of around 100 pg/ml and that this concentration was enough to cause bone growth in culture. Earlier in vitro studies in the literature were performed with significantly higher concentrations (Chen et al., 2006;Jenner et al., 2007;Seemann et al., 2005;Zeng et al., 2007): for example, a stimulatory effect of GDF-5 on the chondrocyte ATDC5 cells was described with dosed of 100 ng/ml (Itoh et al., 2008), 1 mg/ml (Nakamura et al., 1999) or 5 nM (Sammar et al., 2004), which are 100-1000-fold greater than the present study.
Nutritional-induced catch up growth is usually associated with a rapid increase in fat mass suggesting a role for adipocytes in the nutritional-induced growth stimulation. Involvement of GDF-5 in adipocyte-induced growth stimulation is supported by our findings on its increased level in refed animals compared to food restricted ones. Increase in food consumption, body weight and leptin levels were previously reported by us in both mice and rats (Even-Zohar et al., 2008;), but the observation on the increase in GDF-5 is new. Differences in the level of the GDF-5 in the blood between mice and rats may be due to difference in the species tested, but in both of them the increase was significant.
This study was limited by our failure to complete the SILAC analysis. The requirement for serum in the CM may sometimes be solved by the use of serum-free medium or by short-term serum-free starvation of the cells. However, in our case, for the cells to secrete the ''metatarsal growth factor'', they had to be grown with at least 5% of FBS. We performed SILAC followed by Proteominer Õ purification and MS/MS analysis essentially as described by Molina et al. (2009). The presence of heavy amino acids in the peptides proved the cellular origin of the corresponding highly abundant proteins as well as the adipogenic nature of the differentiated cells, but failed to identify low abundant proteins owing to the high concentration of serum albumin.
Using an expression array we identified 19 growth factors which expression was increased in adipocytes by at least twofold; of these we analyzed the two most differentially expressed factors: GDF-5 and leptin. We confirmed the effect of GDF-5 by showing reduced growth in the presence of antibodies directed against GDF-5 in the CM. The contribution of additional factors identified by the expression array, as well as those that may have been missed due to the design of the array, may also be important.

Conclusions
This study shows that GDF-5, a skeletal growth factor, is produced and secreted by adipocytes in culture and stimulates the growth of metatarsals in vitro. The level of GDF-5 is also increased under conditions of nutritional catch up growth in vivo. These results may shed new light on cross communication between these closely associated systems, showing that adipocytes may stimulate bone growth. The results add a new potential mediator to the obesity-growth link and may suggest an additional explanation to the growth without GH phenomenon. Further studies are required to investigate the clinical relevance of our findings: whether adipocytes secrete GDF-5 in vivo in humans and the manner by which this stimulates growth. As the clinical toolbox to treat children with short stature is currently very limited, finding additional growth stimulating factors is of utmost importance; our results may suggest that GDF-5 should be further explored as a novel therapeutic agent to treat children with growth abnormalities.